Gene therapy for cerebrovascular disorders

ABSTRACT

By introducing hepatocyte growth factor (HGF) gene and/or vascular endothelial growth factor (VEGF) gene into the subarachnoid space in humans, cerebrovascular disorders such as cerebrovascular obstruction, cerebral infarction, cerebral thrombosis, cerebral embolism, stroke, cerebral bleeding, moyamoya disease, cerebrovascular dementia, and Alzheimer&#39;s dementia can be effectively treated or prevented.

TECHNICAL FIELD

The present invention relates to novel gene therapy agents for treatingor preventing cerebrovascular disorders, and novel methods foradministration of said gene therapy agents. More preferably, the presentinvention relates to therapeutic or preventive agents forcerebrovascular disorders comprising hepatocyte growth factor (HGF) geneand/or vascular endothelial growth factor (VEGF) gene as an activeingredient, or novel administration methods comprising administeringsaid therapeutic or preventive agents to the subarachnoid space.

BACKGROUND ART

Cerebral obstructive diseases, moyamoya disease and the like caused byatherosclerosis in the cerebral artery often result in chronic reductionin cerebral blood flow. This state may lead not only to the subsequentcerebral ischemic events but also to neuropathological changes includingdementia (Stroke 25:1022-1027, Stroke 29:1058-1062 (1998)); Stroke24:259-264 (1993); Ann. N. Y. Acad. Sci. 695:190-193 (1993)). However,no effective methods for improving the reduced blood flow in thesecerebrovascular disorders have been established yet. It is known that inischemic attacks, active angiogenesis takes place specifically at theperipheral regions of the ischemia, and this is involved in prolongedsurvival in humans (Stroke 25:1794-1798 (1994)). Thus, angiogenesis hasbeen considered to play an important role in recovery from cerebralischemia and prevention of future attacks.

The development of new blood vessels and angiogenesis are triggeredconcurrently with the activation of the endothelial cell. A growthfactor that has been shown not only to stimulate angiogenesis in vivo,but also to be mitogenic in vitro to the endothelial cell is called“angiogenic growth factor.”

The therapeutic involvement of angiogenic growth factor was firstdescribed in literature by Folkman et al. (N. Eng. J. Med. 285:1182-1186(1971)). Subsequent research confirmed that recombinant angiogenicfactor such as fibroblast growth factor (FGF) family (Science257:1401-1403 (1992); Nature 362:844-846 (1993)), endothelial growthfactor (J. Surg. Res. 54:575-583 (1993)), and vascular endothelialgrowth factor (VEGF) may be used to promote and/or enhance thedevelopment of collateral circulation shunt in animal models ofmyocardial and hindlimb ischemia (Circulation 90:II-228-II-234 (1994)).Furthermore, the present inventors have found that HGF acts as aendothelium-specific growth factor as does VEGF (J. Hypertens.14:1067-1072 (1996)).

A strategy as described above that employs angiogenic growth factor totreat vascular disorders is called “therapeutic angiogenesis.” Morerecently, the strategy has been applied to ischemic diseases in humans.However, the effectiveness of the strategy in cerebral ischemia has notbeen known so far.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that exhibitsmitogenic, motility promoting, and morphogenic activity on a variety ofcells (Nature 342:440-443 (1989)).

Effects of HGF on the brain has been reported as follows. Thus, it isknown that HGF in combination with c-Met/HGF receptor of a transmembranetyrosine kinase is expressed at various regions in the brain, and theoperative linkage of HGF and c-Met enhances the survival of neurons inthe primary culture of hippocampus, and induces neutrite elongation inthe development in vitro of neurons (J. Cell. Biol. 126:485-494 (1994);Japanese Unexamined Patent Publication (Kokai) No. 7-89869). Recently,it has been reported that HGF is induced in neurons in ischemia (BrainRes. 799:311-316 (1998)), that recombinant HGF has a neuroprotectiveeffect on delayed neuronal death after ischemia in the hippocampus, andthat the continuous injection of recombinant HGF into the brain waseffective in reducing the size of infarction (J. Cereb. Blood FlowMetab. 18:345-348 (1998)). These findings suggest that HGF acts as animportant neurotrophic factor in cerebral ischemia.

On the other hand, vascular endothelial growth factor (VEGF) is adimeric glycoprotein mitogenic to the endothelial cell and has anability of enhancing vascular permeability. VEGF has a direct andspecific mitogenic effect on the endothelial cell (Biochem. Biophys.Res. Commun. 161:851-858 (1989)). The binding sites of VEGF includingtyrosine kinase receptor Flt, Flk-1, and KDR occur on the endothelialcell but not on other cell types, thereby limiting the effect of VEGF tothe endothelial cell.

With respect to the effect of VEGF on the brain, it has been reportedthat VEGF in the central nervous system is rapidly induced by ischemicdisorders in the brain (Mol. Cell. Biol., 16:4604-4613 (1996)), and thatthe administration of recombinant VEGF to the brain surface effectivelyreduced the amount of infarction (J. Cereb. Blood Flow Metab. 18:887-895(1998)). Details thereof has not been known, however.

In another aspect, in addition to the above-mentioned actions of HGF andVEGF, these factors are potent angiogenic growth factors as mentionedabove (J. Cell. Biol. 119:629-641 (1992)); Biochem. Biophys. Res.Commun. 161:851-858 (1989)). Ischemic attacks are known to give rise toactive angiogenesis in the periphery of ischemia, which is related toprolonged survival of humans (Stroke 25:1794-1798 (1994)). Thus,angiogenesis is thought to play an important role in recovery fromcerebral ischemia and in prevention of future attacks. However, it isnot known whether therapeutic angiogenesis using recombinant HGF or VEGFis actually feasible for cerebral ischemia etc. Furthermore, recombinantangiogenic growth factors rapidly disappear from the brain and thusrequire continuous injection into the brain, which procedure is ratherdangerous and impractical in the clinical settings. Thus, it would bereasonable if the technique of gene introduction is used to express andsecrete angiogenic growth factors in ischemic brain and its periphery ona continual basis. There are no examples so far in which HGF gene orVEGF gene has been applied (gene therapy) to ischemic disorders in thebrain, and possibly because of its unique feature of the brain tissue,there are no suggestions made on the applicability thereof.

DISCLOSURE OF THE INVENTION

The present invention relates to novel gene therapy agents for treatingor preventing cerebrovascular disorders, and novel methods foradministration of said gene therapy agents. More preferably, the presentinvention relates to novel agents for treating and preventingcerebrovascular disorders comprising hepatocyte growth factor (HGF) geneand/or vascular endothelial growth factor (VEGF) gene as an activeingredient, or novel administration methods comprising administeringsaid therapeutic or preventive agents to the subarachnoid space.

The present inventors investigated in vivo whether the introduction ofHGF gene and VEGF gene can induce angiogenesis on the surface of anischemic brain. As a result, we have revealed that: (a) after thetransfection of HGF gene or VEGF gene, these proteins are detected inthe brain over a prolonged period of time, (b) therapy with HGF gene orVEGF gene transfection can induce angiogenesis on the surface of anischemic brain, (c) the transfection of HGF gene or VEGF gene iseffective In treating reduced blood flow in the brain caused by vascularobstruction, and (d) the therapy is also useful when performed beforeobstruction. Furthermore, we have also demonstrated that theintroduction of these genes can be more effectively attained by a novelmethod of administration i.e., introduction into subarachnoid space.

In addition, the present inventors have found that delayed neuronaldeath due to ischemia in the hippocampus CA-1 region can be suppressedby the introduction of HGF gene.

Based on the foregoing findings, the present invention was completed.

Thus, the present invention provides the inventions described in thefollowing (1) to (23).

(1) A therapeutic and preventive agent for cerebrovascular disorderscomprising HGF gene and/or VEGF gene as an active ingredient;

(2) The therapeutic or preventive agent in the above (1) whereincerebrovascular disorders are cerebrovascular obstruction, cerebralinfarction, cerebral thrombosis, cerebral embolism, stroke, cerebralbleeding, moyamoya disease, cerebrovascular dementia, Alzheimer'sdementia, and sequelae of cerebral bleeding or cerebral infarction;

(3) A therapeutic or preventive agent for reduced blood flow in thebrain comprising HGF gene and/or VEGF gene as an active ingredient;

(4) A promoting agent for angiogenesis in the brain comprising HGF geneand/or VEGF gene as an active ingredient;

(5) A suppressing agent for neuronal death in the brain comprising HGFgene as an active ingredient;

(6) The suppressing agent of the above (5) wherein neuronal death in thebrain is delayed neuronal death caused by cerebral ischemia;

(7) A suppressing agent for apoptosis of nerve cells in the braincomprising HGF gene as an active ingredient;

(8) The agent in any of the above (1)-(7) which comprises HGF geneand/or VEGF gene as an active ingredient and which is to be used incombination with HGF protein and/or VEGF protein;

(9) The agent of the above (8) which comprises HGF gene as an activeingredient and which is to be used in combination with HGF protein;

(10) The agent in any of the above (1)-(9) wherein HGF gene and/or VEGFgene are in the form of HVJ-liposome;

(11) The agent in any of the above (1)-(10) to be administered into thesubarachnoid space;

(12) A method of producing the agent in any of the above (1)-(11)comprising blending HGF gene and/or VEGF gene with a pharmaceuticallyacceptable solvent;

(13) A therapeutic or preventive method for cerebrovascular disorderscomprising introducing HGF gene and/or VEGF gene into humans;

(14) A therapeutic or preventive method for reduced blood flowcomprising introducing HGF gene and/or VEGF gene into humans;

(15) A method of promoting cerebral angiogenesis comprising introducingHGF gene and/or VEGF gene into humans;

(16) A method of suppressing neuronal death in the brain comprisingintroducing HGF gene into humans;

(17) A method of suppressing apoptosis of nerve cells in the braincomprising introducing HGF gene into humans;

(18) The method in any of the above (13)-(17) comprising administeringHGF gene and/or VEGF gene into the subarachnoid space in humans;

(19) The method in any of the above (13)-(18) comprising administeringHGF protein and/or VEGF protein together with the introduction of HGFgene and/or VEGF gene;

(20) The method in the above (19) comprising administering HGF proteintogether with the introduction of HGF gene;

(21) Use of HGF gene and/or VEGF gene in the manufacture of atherapeutic or preventive agent for cerebrovascular disorders;

(22) Use of HGF gene and/or VEGF gene in the manufacture of atherapeutic or preventive agent for reduced blood flow in the brain;

(23) Use of HGF gene and/or VEGF gene in the manufacture of a promotingagent for angiogenesis in the brain;

(24) Use of HGF gene in the manufacture of a suppressing agent forneuronal death in the brain; and

(25) Use of HGF gene in the manufacture of a suppressing agent forapoptosis of nerve cells in the brain.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a photograph of morphology of an organism exhibiting theexpression of β-gal (β-galactosidase) on the brain surface. Bottom, theinjection of HVJ-liposome (1 ml) into the internal carotid artery;middle, the injection of HVJ-liposome (100 μl) Into the cisterna(subarachnoid space); upper, the injection of HVJ-liposome (20 μl) intothe lateral ventricle. n=4 for each group.

FIG. 2 is a photograph of morphology of an organism exhibiting theexpression of β-gal (β-galactosidase) in the brain. Left, the injectioninto the internal carotid artery; middle, the injection into the lateralventricle; right, injection into the cisterna (subarachnoid space).

FIG. 3 is a graph showing the in vivo expression of human HGF protein inthe rat cerebrospinal fluid by an ELISA method. In the figure, UTrepresents the rats treated with an expression vector containing no HGFgene, 7d represents the rats on day 7 after the introduction of HGFgene, and 14d represents the rats on day 14 after the introduction ofHGF gene. In the figure also, − represents the absence of obstruction,and + represents the presence of obstruction in the carotid artery. Theordinate represents the concentration of HGF (ng/ml). **: P<0.01 for UT.n=4 for each group.

FIG. 4 is a graph showing the in vivo expression of human VEGF proteinin the rat cerebrospinal fluid by an ELISA method. In the figure, UTrepresents the rats treated with an expression vector containing no VEGFgene, 7d represents the rats on day 7 after the introduction of VEGFgene, and 14d represents the rats on day 14 after the introduction ofVEGF gene. In the figure also, − represents absence of obstruction inthe carotid artery, and + represents the presence of obstruction. Theordinate represent the concentration of VEGF (pg/ml). **: P<0.01 for UT.n=4 for each group.

FIG. 5 is a microphotograph showing the result of cytohistochemicalstaining of the endothelial cell in the brain and in its peripherybefore and 7 days after the transfection of HGF gene. A (upper left), abrain transfected with a vector (expression vector containing no HGFgene) without obstructing the carotid artery; B (upper right), a braintransfected with HGF gene without obstructing the carotid artery; C(bottom left), a brain transfected with a vector on day 7 after theobstruction of the carotid artery; D (bottom right), a brain transfectedwith HGF gene on day 7 after the obstruction of the carotid artery. n=4for each group.

FIG. 6 is a graph showing changes in cerebral blood flow with timemeasured by a laser Doppler imager (LDI). In the figure, pre representsbefore obstruction, post represents after the obstruction of the carotidartery, 7d represents 7 days after obstruction, and 14d represents 14days after obstruction. The ordinate (FLUX) represents an mean cerebralperfusion. Relative to pre, *P<0.05, **P<0.01. n=6 for each group.

FIG. 7 is a graph showing CBF measured by LDI on day 7 after theobstruction of the carotid artery. In the figure, UP represents the ratstreated with an expression vector, RC represents the rats treated withrecombinant HGF (200 μg), GENE represents the rats treated with HGF gene(10 μg), GENE&RC represents the rats treated with recombinant HGF (200μg) and HGF gene (10 μg), and GENE in VEGF represents the result of therats treated with VEGF gene (20 μg/ml). The ordinate (FLUX) is an meancerebral perfusion. Relative to pre, *P<0.05, **P<0.01. n=6 for eachgroup.

FIG. 8 is a graph showing CBF measured by LDI before and immediatelyafter the obstruction of the carotid artery. In the figure, prerepresents before the obstruction of the carotid artery of the controlrats, post represents immediately after the obstruction of the carotidartery, HGF represents the result of the rats immediately after carotidartery obstruction that were subjected to HGF transfection 7 days beforearterial obstruction, and VEGF represents the result of the ratsimmediately after carotid artery obstruction that were subjected to VEGFtransfection 7 days before arterial obstruction. Relative to post,**P<0.01. n=5 for each group.

FIG. 9 is a microphotograph showing the expression of β-gal(β-galactosidase) on the brain surface (brain surface in the figure) andin the hippocampus CA-1 region (CA1 in the figure).

FIG. 10 is a microphotograph showing the result in which delayedneuronal death was observed in the hippocampus CA-1 region by ischemicstimulation of the bilateral carotid arteries. In the figure, Sham ope.7 days represents the result on day 7 of the control (surgicallytreatment only without ischemic stimulation), and Vehicle (4 days, 7days) represents the result on day 4 and 7 after ischemia of thebilateral carotid arteries, respectively.

FIG. 11 is a microphotograph showing the result in which delayedneuronal death in the hippocampus CA-1 region was suppressed by theintroduction of HGF gene or recombinant HGF protein before and afterischemic stimulation of the bilateral carotid arteries. In the figure,Post HGF gene (4 days, 7 days) represents the result on day 4 and day 7in which HGF gene was introduced immediately after ischemia of thebilateral carotid arteries, Pre HGF gene 7 days represents the result onday 7 in which HGF gene was introduced immediately before ischemia ofthe bilateral carotid arteries, and r-HGF 7 days represents the resulton day 7 in which recombinant HGF protein was introduced immediatelyafter ischemia of the bilateral carotid arteries.

FIG. 12 is a graph showing the result in which the density of nervecells in the hippocampus CA-1 region was measured by staining the livenerve cells. In the figure, the ordinate represents the cell density(live nerve cell count/mm). Sham in the abscissa represents the resultof the control (no ischemic stimulation), vehicle represents the resultof ischemia of the bilateral carotid arteries, PostG represents theresult in which HGF gene was introduced immediately after ischemia ofthe bilateral carotid arteries, PreG represents the result in which HGFgene was introduced before ischemia of the bilateral carotid arteries,and PostR represents the result in which recombinant HGF protein wasintroduced after ischemia of the bilateral carotid arteries. Relative tovehicle, *P<0.05, **P<0.01, and ***P<0.001.

FIG. 13 is a graph showing the result in which HGF gene was introducedafter ischemia of the bilateral carotid arteries and then the proteinconcentration of HGF in the cerebrospinal fluid 7 days later wasmeasured by an ELISA method. In the figure, the ordinate represents theprotein concentration (ng/ml) of HGF, post HGF on the abscissarepresents the result of introduction of HGF gene, and sham representsthe result of the control (no ischemic stimulation). N.D. represents theresult not detected.

FIG. 14 is a microphotograph showing the result in which the expressionof C-Met in the hippocampus CA-1 region was analyzed by animmunostaining method.

FIG. 15 is a microphotograph showing the result in which the nerve cellsthat had apoptosis in the hippocampus CA-1 region were stained by theTUNEL method. In the figure, DND 7 days represents the nerve cells thathad delayed neuronal death on day 7 after ischemia of the bilateralcarotid arteries, Post HGF gene 7 days represents the result on day 7after HGF gene was introduced immediately after ischemia of thebilateral carotid arteries, and Pre HGF gene 7 days represents theresult on day 7 after HGF gene was introduced immediately beforeischemia of the bilateral carotid arteries.

FIG. 16 is a microphotograph showing the result in which the expressionof Bcl-xL in the hippocampus CA-1 region was analyzed by animmunostaining method. In the figure, sham. represents the result of thecontrol (no ischemic stimulation), post HGF (4 days, 7 days) representsthe result on day 4 and day 7 after HGF gene was introduced immediatelyafter ischemia of the bilateral carotid arteries.

FIG. 17 is a microphotograph showing the result in which HSP70expression In the hippocampus CA-1 region on day 7 after theintroduction of HGF gene immediately after ischemia of the bilateralcarotid arteries was analyzed by an immunostaining method.

FIG. 18 is a microphotograph showing the result in which HSP70expression in the hippocampus CA-1 region was analyzed by animmunostaining method. In the figure, Sham. represents the result of thecontrol (no ischemic stimulation), and Post HGF 7 D represents theresult on day 7 after the introduction of HGF gene immediately afterischemia of the bilateral carotid arteries.

BEST MODE FOR CARRYING OUT THE INVENTION

As used herein “HGF gene” means a gene that can express HGF (HGFprotein). Specifically, there can be mentioned one in which cDNA of HGFas described in Nature 342:440 (1989); Patent Publication No., 2777678;Biochem. Biophys. Res. Commun. 163:967 (1989); and Biochem. Biophys.Res. Commun. 172:321 (1990) was integrated into a suitable expressionvector (nonviral vector, viral vector) as described below. The basesequence of cDNA encoding HGF has been described in the above literatureand also been registered at databases such as Genbank. Thus, based onsuch sequence information, a suitable DNA portion is used as a PCRprimer; for example, by performing a RT-PCR reaction on mRNA derivedfrom the liver or leukocytes, cDNA of HGF can be cloned. Such cloningcan easily be performed by a person skilled in the art according to abasic textbook such as Molecular Cloning 2nd Ed., Cold Spring HarborLaboratory Press (1989).

Furthermore, the HGF gene of the present invention is not limited tothose described above, but any gene may be used as the HGF gene of thepresent invention as long as the protein expressed by said gene can actin virtually the same manner as HGF. Thus, from among 1) DNA thathybridizes to said cDNA under a stringent condition and 2) DNA encodinga protein comprising an amino acid sequence in which one or a pluralityof (preferably several) amino acids have been substituted in, deletedfrom, and/or added to the amino acid sequence of the protein encoded bysaid cDNA and the like, those that encode a protein having an action asHGF are encompassed in the category of HGF gene of the presentinvention. DNA in the above 1) and 2) may be easily obtained bysite-directed mutagenesis, a PCR method, or a standard hybridizationmethod and the like, and specifically they may be performed withreference to a basic textbook such as the above Molecular Cloning etc.

As used herein “VEGF gene” means a gene that can express VEGF (VEGFprotein). Thus, there can be illustrated one integrated into a suitableexpression vector (nonviral vector, viral vector) as described below. Byselective splicing of VEGF gene at transcription in humans, the presenceof 4 subtypes (VEGF121, VEGF165, VEGF189, VEGF206) have been reported(Science 219:983 (1983); J. Clin. Invest. 84:1470 (1989); Biochem.Biophys. Res. Commun. 161:851 (1989)). According to the presentinvention, any of these VEGF genes can be used, but from the viewpointof being biologically most potent, VEGF165 gene is most preferred.Furthermore, as in the case of the above HGF, modified versions of theseVEGF genes are encompassed in the category of the VEGF gene of thepresent invention as long as they encode a protein having an activity asVEGF.

Said VEGF gene, as in the case of HGF gene, may be easily cloned by aperson skilled in the art based on sequences as previously described(for example, Science 246:1306 (1989)) and sequence informationregistered in databases, and their modification can also be easilyperformed.

According to the present invention, it was demonstrated for the firsttime that cerebrovascular disorders can be treated or prevented with HGFgene or VEGF gene. Thus, the present invention revealed, for the firsttime, that (a) after the transfection of HGF gene or VEGF gene, theseproteins are detected in the brain over a prolonged period of time, (b)by treatment using HGF gene or VEGF gene transfection, angiogenesis canbe induced on the surface of an ischemic brain, (c) the transfection ofHGF gene or VEGF gene is effective in treating reduced blood flow in thebrain caused by obstruction in the blood vessels, and (d) this treatmentmethod is also effective when performed before obstruction. Thus, HGFgene and VEGF gene may be effectively used as a therapeutic orpreventive agent for various cerebrovascular disorders such as disordersresulting from cerebral ischemia, disorders associated with reducedblood flow in the brain, disorders for which improvement is expected bypromoting angiogenesis in the brain, and the like.

Specifically they are effectively used as therapeutic or preventiveagents (hereinafter, the therapeutic or preventive agents of the presentinvention are simply designated as gene therapy agents) forcerebrovascular obstruction, cerebral infarction, cerebral thrombosis,cerebral embolism, stroke (including subarachnoid bleeding, transientcerebral ischemia, cerebral atheroscrelosis), cerebral bleeding,moyamoya disease, cerebrovascular dementia, Alzheimer's dementia,sequelae of cerebral bleeding or cerebral infarction, and the like.

Furthermore, the present inventors have found that delayed neuronaldeath due to ischemia in the hippocampus CA-1 region is suppressed bythe introduction of HGF gene, that is. HGF gene has an effect ofsuppressing neuronal death in the brain. We have also demonstrated thatthis effect is based on the c-Met-mediated apoptosis-suppressing effectof nerve cells.

The hippocampus CA-1 region as used herein is a region that is denselypopulated with nerves and a region that is susceptible to neuronal deathby cerebral ischemia. Such HGF gene has been found to be able to treatand prevent cerebrovascular disorders based on the both of theangiogenic effect (suppression of reduced blood flow) and the nerve cellprotective effect.

Since HGF gene has c-Met-mediated nerve cell protecting effect asdescribed above, it can be effectively used as a therapeutic orpreventive agent for neurodegenerative diseases such as Alzheimer'sdisease, Alzheimer's senile dementia, amyotrophic lateral sclerosis, orParkinson's disease.

In accordance with the present invention, HGF gene and VEGF gene may beused alone or in combination with each other. They can also be used incombination with the gene of other vascular endothelial growth factors.Furthermore, HGF gene and/or VEGF gene may be used in combination withHGF protein and/or VEGF protein. Preferred are a combination of HGF geneand HGF protein or of VEGF gene and VEGF protein, more preferably of HGFgene and HGF protein. See Example 4 below for details.

HGF protein as used herein may be obtained by any method as long as ithas been purified to the extent it may be usable as a pharmaceuticaldrug. Commercially available products (for example Toyoboseki k. k.,Code No. HGF-101, etc.) may also be used. cDNA of HGF obtained bycloning mentioned above is inserted into any suitable expression vector,which is introduced into a host cell to obtain a transformant, from theculture supernatant of which transformant may be obtained recombinantHGF protein of interest (see, for example, Nature 342:440 (1989): PatentPublication No. 2777678). VEGF protein can also be obtained in a similarmanner.

Then, a method of gene introduction, form of introduction, amount to beintroduced and the like for use in gene therapy of the present inventionare explained.

When a gene therapy agent comprising the above gene as an activeingredient is to be administered to patients, the dosage regimens areroughly divided into two: a case in which a nonviral vector is used, anda case in which a viral vector is used. The methods of preparation andadministration thereof are explained in detail in experimental manuals(Separate volume of Experimental Medicine, Basic Technology in genetherapy, Yodosha (1996); Separate volume of Experimental Medicine,Experimental Methods in Gene Introduction and Expression Analysis,Yodosha (1997); Handbook for Development and Research of Gene Therapy,edited by Japan Society of Gene Therapy, NTS (1999)). This will beexplained in specific terms below.

A. When a Nonviral Vector is Used

Using a recombinant expression vector in which a gene of interest hasbeen integrated into a commonly used gene expression vector may be usedto introduce the gene of interest into cells or tissue by the followingmethod etc.

As a method of gene introduction into cells, there can be mentioned thelipofection method, the calcium phosphate co-precipitation method, theDEAE-dextran method, direct DNA introduction methods using micro glasstubes, and the like.

As a method of gene introduction into the tissue, a recombinantexpression vector may be incorporated into the cell by subjecting any ofa method of gene introduction with internal type liposome, a method ofgene introduction with electrostatic type liposome, the HVJ-liposomemethod, the improved HVJ-liposome method (HVJ-AVE liposome method), thereceptor-mediated gene introduction method, a method of introducing DNAmolecules together with carriers (metal particles) by a particle gun, amethod of directly introducing naked-DNA, a method of introduction withpositively-charged polymers and the like.

Among them, HVJ-liposome is a fusion product prepared by enclosing DNAinto liposome made of lipid bilayer, which was fused to inactivatedSendai virus (Hemagglutinating virus of Japan: HVJ). The HVJ-liposomemethod is characterized by a very high fusing activity with the cellmembrane compared to the conventional liposome method, and is apreferred mode of introduction. For the method of preparingHVJ-liposome, see, for details, the literature (Separate volume ofExperimental Medicine, Basic Technology in gene therapy, Yodosha (1996);Experimental Methods in Gene Introduction and Expression Analysis,Yodosha (1997); J. Clin. Invest. 93:1458-1464 (1994); Am. J. Physiol.271:R1212-1220 (1996)) and the like, and experimental examples describedbelow for details. As HVJ, the Z strain (available from ATCC) ispreferred, but other HVJ strains (for example, ATCC VR-907 and ATCCVR-105) may also be used.

Furthermore, the method of directly introducing naked-DNA is the mostsimple method among the methods described above, and in this regard apreferred method of introduction.

Expression vectors as used herein may be any expression vectors as longas they permit the expression in vivo of the gene of interest, andinclude, for example, expression vectors such as pCAGGS (Gene108:193-200 (1991)), pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stratagene)and the like.

B. When a Viral Vector is Used

Representative methods use, as viral vectors such as recombinantadenovirus, retrovirus and the like. More specifically, the gene ofinterest can be introduced into DNA virus or RNA virus such asdetoxified retrovirus, adenovirus. adeno-associated virus, herpesvirus,vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40,human immunodeficiency virus (HIV) and the like, which is then infectedto the cell to introduce the gene into the cell.

Among the above viral vectors, the efficiency of infection is known tobe the highest with adenovirus than with other viral vectors. In thisregard, it is preferred to use an adenovirus vector system.

As methods of introducing a gene therapy agent into a patient, there arean in vivo method that permits direct introduction of the gene therapyagent into the body, and an ex vivo method in which certain cells areremoved from a human and a gene therapy agent is introduced into saidcells, which are then returned into the body (Nikkei Science, April 1994issue pp. 20-24; Monthly Yakuji, 36(1):23-48 (1994); Supplement toExperimental Medicine 12(15) (1994); Handbook for Development andResearch of Gene Therapy, edited by Japan Society of Gene Therapy, NTS(1999)). According to the present invention, the in vivo method ispreferred.

Sites for administration to patients are selected depending on thedisease, disease state and the like to be treated. For example, inaddition to making a hole directly into the cranium and introducing thegene therethrough, there is administration to the lateral ventricle oradministration to the subarachnoid space. Among them, administration tothe subarachnoid space is a novel and efficient method of administrationthat was disclosed in the present invention. The administration to thesubarachnoid space is desired when it is intended to treat the diseasebased on the original purpose. i.e. when reduced blood flow in the brainis treated by angiogenesis and/or by suppressing neuronal death in thebrain.

Dosage forms may take various forms according to various administrationregimens described above (for example, liquids). When, for example, aninjection containing the gene as an active ingredient is to be used,said injection may be prepared according to a standard method. Forexample, after dissolving in a suitable solvent (a buffer such as PBS,physiological saline, sterile water, etc.), it is filter-sterilized withfilter as needed, and then filled into sterilized containers. Commonlyused carriers etc. may be added to the injection. In liposomes such asHVJ-liposome, they may take the form of suspensions, frozenformulations, centrifugation-concentrated frozen formulations and thelike.

In order to facilitate delivery of the gene into the periphery of alesion site, a sustained release preparation (minipellet formulation,etc.) may be prepared and implanted near the affected region, or it canbe administered to the affected area continuously and gradually using anosmotic pump etc.

The content of DNA in the formulation may be controlled as appropriatedepending on the disease to be treated, age and weight of the patient,etc., and usually it is in the range of 0.0001-100 mg, preferably0.001-10 mg, as the DNA of the present invention, which is preferablygiven every few days to every few months.

The present invention will now be specifically explained with referenceto the following examples. It should be noted, however, that the presentinvention is not limited by these examples in any way.

Experiment I.

A Study on Angiogenesis and Effect of Improving Blood Flow in the Brainwith HGF Gene and VEGF Gene

Materials and Experimental Methods

1) Ligation of the Bilateral Carotid Arteries

Male Sprague Dawley rats (350-400 g; Charles River Japan, Atsugi city,Japan) were anesthetized with pentobarbital sodium (50 mg/kg,intraperitoneal), and were allowed to breathe spontaneously duringsurgery. By midline neck incision, the bilateral carotid arteries wereexposed, and were tightly ligated by 2-0 silk.

2) Preparation of HVJ-liposome Complex

The method used to prepare HVJ-liposome is as previously described (J.Clin. Invest. 93:1458-1464 (1994); Am. J. Physiol. 271:R1212-1220(1996)). Briefly, phosphatidyl serine, phosphatidyl choline, andcholesterol were mixed at a weight ratio of 1:4.8:2. Tetrahydrofuran wasremoved by rotary evaporator to allow the lipid mixture (10 mg) todeposit on the side wall of the flask. The dried lipid was hydrated in200 μl of a balanced salt solution (BSS: 137 μM NaCl, 5.4 μM KCl, 10 μMTris-HCl, pH 7.6) having an expression vector in which the gene ofinterest had been inserted. Liposomes in the control group contain anexpression vector having no gene of interest (BSS 200 μl). Liposomeswere prepared by shaking and ultrasonication.

Purified HVJ (Z strain) was inactivated by UV irradiation (110 erg/mm²per second) for 3 minutes immediately prior to use. A liposome mixture(0.5 ml containing 10 mg of lipid) was mixed with HVJ (10,000hemagglutination units in a total volume of 4 ml). The mixture wasincubated at 4° C. for 5 minutes, and then at 37° C. for 30 minuteswhile shaking gently. Free HVJ was removed from the HVJ-liposome bysucrose density gradient centrifugation. The uppermost layer of thesucrose gradient was collected and used. The final concentration ofplasmid DNA was equal to 20 μg/ml when calculated as previously reported(J. Clin. Invest. 93:1458-1464 (1994); Am. J. Physiol. 271:R1212-1220(1996)). The method of preparation has been optimized so as to attainthe maximum transfection efficiency.

3) In vivo Gene Introduction

In order to establish an efficient method of in vivo gene introduction,we have tested three different methods to deliver plasmid that formed acomplex with the HVJ-liposome: 1) direct introduction into the internalcarotid artery, 2) injection into the lateral ventricle, and 3)injection into the cisterna (subarachnoid space).

For the introduction into the internal carotid artery, male SpragueDawley rats (350-400 g) were anesthetized with pentobarbital sodium (50mg/kg, intraperitoneal), and incision was made to the left commoncarotid artery, into which a polyethylene catheter (PE-50, Clay Adams,Parsippany, N.J.) was introduced (Rakugi et al.). The distal region ofthe external carotid artery was isolated for a short time by closingwith ligature temporarily. The HVJ-liposome complex (1 ml) was injectedinto the external carotid artery region. After injection, the injectioncanula was removed and the ligature was loosened to recover blood flowinto the common carotid artery.

For injection into the lateral ventricle, the anesthetized rats wereplaced in a stereotaxic apparatus (Narishige Scientific InstrumentLaboratory, Tokyo, Japan) to expose the cranium. A stainless steelcanula (30 gauge; Becton Dickinson, Franklin Lakes, N.J.) with aspecifically designed Teflon connector (FEP tube, Bioanalytical Systems,West Lafayette, Ind.) was introduced into the left lateral ventricle adpreviously described (Am. J. Physiol. 271:R1212-1220 (1996)). Thestereotaxic coordinate was as follows: behind the bregma, 1.3 mm; sideof the midline, 2.1 mm; and under the cranial surface, 3.6 mm. TheHVJ-liposome complex was injected to the lateral ventricle (20 μl).After the injection of the HVJ-liposome complex, the injection canulawas removed. No behavioral changes such as spasm in the extremities andabnormal movement were observed in any animal that received injection.

For injection into the subarachnoid space, the head of each animal wasfixed at a horizontal position, and the atlantoccipital membrane wasexposed by the midline incision of the occipital bone. A stainless steelcanula (27 gauge; Becton Dickinson, Franklin Lakes, N.J.) was introducedinto the subarachnoid space. The position of the canula was confirmed,and in order to avoid increases in intracranial pressure, 100 μl ofcerebrospinal fluid was removed. Then the HVJ-liposome solution (100 μl:100 μg/ml) was carefully injected into the cisterna (subarachnoid space)over more than one minute. Then the animal was placed with the head downfor 30 minutes. A preventive dosage of antibiotics (30,000 U penicillin)was administered to complete the sterile procedure.

4) Laser Doppler Imaging

Using a laser Doppler imager (LDI), continuous blood flow was recordedfor two weeks after the surgery. The LDI system (Moore Instruments Ltd.,Devon, UK) has a 2 W built-in helium-neon laser in order to generate abeam that continuously scans to a depth of 600 μm of the tissue surfaceof 12×12 cm. During scanning, blood cells moving in the blood systemchange the frequency of the incident light according to the Dopplerprinciple. A photodiode collects the scattered light in the oppositedirection, and thereby variation in the original light strength areconverted into voltage variation in the range of 0-10 V. A perfusionoutput value at 0 V was graduated at 0% perfusion, and 10 V wasgraduated at 100% perfusion. After scanning is complete and thescattered light in the opposite direction is collected from allmeasurement sites, color-coded images showing blood flow are displayedon a television monitor. Perfusion signals are divided into sixdifferent sections, each being displayed as a distinct color. Thereduced blood flow or no perfusion is indicated by dark blue, while themaximum perfusion is displayed as red.

Using the LDI, perfusion at the brain surface was recorded before,immediately after, 7 and 14 days after obstruction. Along the midlineincision on the scalp, a bone window of 12×12 cm was made using anelectric drill. On this bone window, continuous measurement values wereobtained. Color-coded images were recorded, and analysis was performedby calculating mean perfusion values for each rat. In order to considervariables containing ambient light and temperature, calculated values ofperfusion were expressed as ratios of the brain after (ischemia) tobefore (nontreatment).

5) Histopathological Examination

After fixing in a 3% paraformaldehyde/20% sucrose solution for one day,25 μm frozen sections of coronal plane were made for every 100 μm foruse in X-gal staining. The sections were stained with X-gal to identifystained neurons that are expressing β-galactosidase. 25 μm frozensections of coronal plane were made for every 100 μm for use in alkalinephosphatase (ALP) staining. These sections were incubated together withPBS containing 0.3% hydrogen peroxide to decrease endogenous peroxidaseactivity, and then were incubated with primary antibody diluted in PBScontaining 10% equine serum or a lectin at room temperature for 60minutes. After washing three times in a Tris buffered saline containing2% equine serum, the biotin-tagged secondary antibody compatible withthe species and then avidin-biotin peroxidase complex (Vectastain ABCkit, PK6100, Vector laboratories, Burlingame, Calif.) were incubated.Antibody binding was visualized using diaminobenzidine. Primary antibodywas omitted and stained with unrelated immunoglobulin compatible withthe type and class in order to use as a negative control for eachantibody.

6) ELISA Method on HGF and VEGF in the Cerebrospinal Fluid (CSF)

CSF (100 μl) obtained from rats before, 7 and 14 days after theobstruction of the bilateral carotid arteries was used in theexperiments. Rat and human HGFs were determined by an ELISA kit(Institute of Immunology, Tokyo), and human VEGF was also determined byan ELISA kit (R&D systems, Minneapolis, Minn.).

7) Experimental Materials

cDNA of human HGF (U.S. Pat. No. 2777678) was cloned by a standardmethod, which was inserted into an expression vector pcDNA (manufacturedby Invitrogen) and used as human HGF gene.

cDNA of human VEGF165 (Science 246:1306 (1989)) was cloned by a standardmethod, which was inserted into an expression vector pUC-CAGGS and usedas human VEGF gene.

Using a recombinant expression vector in which cDNA of human HGF (U.S.Pat. No. 2,777,678) was inserted into an expression vector pcDNA(manufactured by Invitrogen), Chinese hamster ovary cells (ATCC) orC-127 cells (ATCC) were transfected, and from the culture medium thereofhuman recombinant HGF was purified by a standard method and used.

Based on the above materials and experimental method, the followingExamples 1-4 were performed.

EXAMPLE 1

Effect of the HVJ-liposome Delivery System on In vivo Transfection ofβ-galactosidase Gene

As a gene to be introduced, β-galactosidase gene (manufactured byInvitrogen, concentration in HVJ-liposome: 20 μg/ml) was used to prepareHVJ-liposome as described in the above materials and experimentalmethod.

First, the HVJ-liposome complex was injected directly into the internalcarotid artery and was allowed to reach the brain. However, in theintraarterial injection in the above carotid artery, little expressionin the brain or microvascular endothelial cells was generated on day 3and 7 after the injection (data not shown). Therefore, HVJ-liposome wasinjected into the lateral ventricle and the subarachnoid space. Theinjection of β-galactosidase gene by the HVJ-liposome method gave riseto marked expression of β-gal on day 3 and 7 after the injection (FIG. 1and FIG. 2). When injected into the lateral brain, β-gal expression wasmainly observed in the lateral ventricle and the perichoroidal plexus.In contrast, when injected into the subarachnoid space, β-gal expressionwas observed on the brain surface. The foregoing result revealed thatinjection into the subarachnoid space is better when the reduced bloodflow in the brain is to be treated by angiogenesis.

EXAMPLE 2

In vivo Transfection of HGF Gene and VEGF Gene

In order to understand the effect of introduction of HGF gene and VEGFgene, the protein expression of these molecules in the cerebrospinalfluid (CSF) was determined by an ELISA method (n=4, each group). First,human HGF and VEGF were determined in the CSF of the control rats(treated with an expression vector in which no HGF gene or VEGF genewere introduced) before, 7 and 14 days after the obstruction of thebilateral carotid arteries, and no concentration of these proteins wasdetected (FIG. 3 and FIG. 4).

Next, the concentration of human HGF protein was determined in the CSFof the rats in which HGF gene (concentration in HVJ-liposome: 20 μg/ml)was introduced into the subarachnoid space immediately after the carotidartery obstruction. On day 7 after transfection, human HGF was detectedbut not rat HGF (FIG. 3). There were no marked differences observebetween the rats (1.63±0.16 ng/ml) in which the carotid artery wasobstructed and the rats (1.67±0.29 ng/ml) in which the carotid arterywas not obstructed. Even on day 14 after transfection, human HGF wasdetected (0.40±0.04 ng/ml) (FIG. 3).

In a similar procedure to the above HGF gene, VEGF gene (concentrationin HVJ-liposome: 20 μg/ml) was introduced into the subarachnoid space,and the concentration of human VEGF in the CSF was much lower than HGF(FIG. 4) (day 7; 18.9±2.9 pg/ml for the rats in which the carotid arterywas not obstructed, and 16.8±5.8 pg/ml for the rats in which the carotidartery was obstructed, day 14; 11.7±1.6 pg/ml for the rats in which thecarotid artery was not obstructed, and 9.9±1.5 pg/ml for the rats inwhich the carotid artery was obstructed). The reason for thesedifferences are unknown, but it appears that it is preferred to causeangiogenesis by applying HGF in order to treat chronic reduction inblood flow.

EXAMPLE 3

Angiogenesis on the Brain Surface by HGF Transfection

Using the tissue of the rats that were treated as in Example 2, theeffect of HGF gene introduction in the CNS on angiogenesis wasconfirmed. Thus, by performing histopathological analysis using analkaline phosphatase (ALP) staining that detects vascular endothelialcells, endothelial cells in and around the brain were detected. In therats in which HGF gene was not introduced, ALP-positive cells werelimited to the inside of the brain before and 7 days after theobstruction of the bilateral carotid arteries (A and C in FIG. 5).Interestingly, in the rats in which HGF gene was introduced,ALP-positive cells were observed on the brain surface, and more cellswere observed on the brain surface in the rats in which the bilateralcarotid arteries were obstructed than in the rats in which the bilateralcarotid arteries were not obstructed (B and D in FIG. 5). These resultssuggested that the introduction of HGF gene caused angiogenesis inparticular on the brain surface in an ischemic state.

EXAMPLE 4

Cerebral Blood Flow (CRF) in the Rat Measured by LDI

CBF in the rat was measured before and after the obstruction of thebilateral carotid arteries. First, changes in CBF of the rats in whichgene was not introduced were analyzed before, immediately after, 7 and14 days after obstruction. As expected, CBF decreased immediately afterobstruction of the bilateral carotid arteries, and gradually increasedwith time (FIG. 6). However, CBF was markedly lower on day 7 and 14after obstruction compared to the non-treated rats (FIG. 6).

Next, the rats treated with recombinant HGF (200 μg), HGF gene(concentration in HVJ-liposome: 20 μg/ml), and a combination ofrecombinant HGF and HGF gene were measured. The HGF gene and recombinantHGF were injected to the subarachnoid space in a manner similar to thatin Examples 2 and 3. Each treatment was performed 10 minutes aftercarotid artery obstruction. In the rats treated with recombinant HGF, nomarked increases in CBF were observed compared to the control rats(control: 886.1±99.6, recombinant HGF: 985.5±142.4) (FIG. 7). However,in the treatment with HGF gene introduction, CBF showed a markedincrease on day 7 after obstruction (1214.5±145.1). Furthermore, in therats treated with a combination of recombinant HGF and geneintroduction, unexpectedly, CBF was much higher on day 7 compared togene introduction alone (1490.3±197.9). These results demonstrated thatangiogenesis by the introduction of HGF gene improves chronic reductionin cerebral blood flow, and that the combination of the gene andrecombinant HGF is the most effective when treated after arterialobstruction.

On the other hand, since VEGF gene also enhanced CBF (1122.8±265.3)(FIG. 7), VEGF gene was also shown to be effective in improving reducedblood flow in the brain.

Next, the effectiveness of the treatment was investigated when it wasperformed before arterial obstruction. Interestingly, the treatment withHGF gene or VEGF gene before arterial obstruction prevented reduction inCBF due to carotid artery obstruction (control: 459.4±97.4, HGF:796.8±204, VEGF: 737.6±211.5) (FIG. 8). These results indicate that whendelivered before ischemia, the introduction of HGF gene and VEGF gene iseffective in preventing reduced blood flow due to arterial obstruction.

Experiment II.

Study on the Suppressive Effect of Neuronal Death in the Brain by HGFGene

Experimental Method

The HVJ-liposome complex containing human HGF gene and human recombinantHGF used in the experiment were prepared in the same manner as in theabove Experiment I.

In the experiment, male Mongolian gerbils (weight: 50-70 g) were used.The animals were bred in a room of which temperature was maintained at24° C. and water and the feed were given ad libitum. The Mongoliangerbils were divided into five groups. “sham”: the control group (groupwith no ischemic stimulation), “vehicle”: the group with 5-min ischemiaof the bilateral carotid arteries, “post G”: the group with HGF geneintroduction after 5-min ischemia of the bilateral carotid arteries,“pre G”: the group with HGF gene introduction before 5-min ischemia ofthe bilateral carotid arteries. “post R”: the group with one-timeadministration of recombinant HGF gene after 5-min ischemia of thebilateral carotid arteries. Wearing a face mask, anesthesia of 3%halothane was performed, and maintained at a mixed air of 1.5%halothane, 20% oxygen, and 80% nitrogen. Body temperature (thetemperature of the rectum) was always monitored to maintain at around37° C. using a heat pad. After the bilateral carotid arteries wereexposed, blood flow was completely blocked using a blood vessel clip for5 minutes. Thereafter, the clip was released to restore blood flow.Immediately before or immediately after the surgical treatment, humanHGF gene (20 μg) was introduced from the subarachnoid space to thecerebrospinal cavity using the HVJ-liposome method. Recombinant HGF (30μg) was given from the subarachnoid space to cerebrospinal cavityimmediately after the surgical treatment. After the surgery also, thecage was maintained at 37° C. to wait for recovery to occur. The controlgroup was treated in the same manner as in the other groups except bloodflow blocking. On day 4 and 7 after ischemia, the brain was extractedand the sections were HE stained, TUNEL stained, and immunostainedbefore performing histopathological analysis. The concentration of HGFin the cerebrospinal fluid was measured using a human HGF ELISAanalysis.

Based on the above experimental method, the following Example 5 wasperformed.

EXAMPLE 5

Suppression of Neuronal Death in the Hippocampus CA-1 Region by HGF GeneTransfection

Using normal Mongolian gerbils, the introduction of gene from thesubarachnoid space to the cerebrospinal cavity by the HVJ-liposomemethod was confirmed. When β-galactosidase gene was introduced and thesections of the brain were β-gal stained, gene expression was observedon the brain surface and the hippocampus CA-1 region (FIG. 9).

By ischemia for 5 minutes at the bilateral carotid arteries, delayedneuronal death was observed in the hippocampus CA-1 region of the brain(FIG. 10, the vehicle group). In contrast, the administration of HGFgene (the PreG group and the PostG group) or recombinant HGF (the PostRgroup) significantly suppressed delayed neuronal death (FIG. 11 and FIG.12). When HGF concentration in the cerebrospinal fluid of the PostGgroup was measured by an ELISA method, HGF expression was observed evenafter 7 days (FIG. 13). Thus, HGF was found to be effective insuppressing delayed neuronal death due to cerebral ischemia.

When the expression site of a HGF receptor, c-Met, was investigated byan immunostaining method, expression was observed in the CA-1 regionindicating that HGF signals are transmitted through c-Met (FIG. 14).

Furthermore, when nerve cells that had apoptosis in the CA-1 region werestained by the TUNEL method, apoptosis of nerve cells was observed inabundance in the vehicle group (FIG. 15). In contrast, little apoptosiswas detected in the HGF gene administration group (the PreG group andthe PostG group) (FIG. 15). Thus, the administration of HGF gene wasthought to suppress apoptosis of nerve cells. In order to investigatethe mechanism of suppression, expression in the CA-1 region of Bcl-xland HSP70 having a apoptosis-suppressing effect was examined byimmunostaining. The expression of Bcl-xL is shown in FIG. 16, and thatof HSP70 is shown in FIG. 17 and FIG. 18. Expression of both proteinswas confirmed in nerve cells by the administration of HGF gene. Theforegoing revealed that the administration of HGF gene induces theexpression of Bcl-xL and HSP70 and suppresses the apoptosis of nervecells.

INDUSTRIAL APPLICABILITY

In accordance with the present invention, there may be provided noveltherapeutic or preventive agents for cerebrovascular disorderscomprising HGF gene and/or VEGF gene as an active ingredient, and noveladministration methods comprising administering said therapeutic orpreventive agents to the subarachnoid space.

1. A therapeutic or preventive method for cerebrovascular disorderscomprising introducing into a human subject a polynucleotide encodinghepatocyte growth factor (HGF) and/or a polynucleotide encoding vascularendothelial growth factor (VEGF) in the form of hemagglutinating virusof Japan (HVJ)-liposomes by direct injection into the subarachnoid spaceof said subject thereby treating or preventing said cerebrovasculardisorders.
 2. A therapeutic or preventive method for treating orpreventing reduced blood flow comprising introducing into a humansubject a polynucleotide encoding HGF and/or a polynucleotide encodingVEGF in the form of HVJ-liposomes by direct injection into thesubarachnoid space of said subject thereby treating or preventing saidreduced blood flow.
 3. A method of promoting cerebral angiogenesiscomprising introducing into a human subject a polynucleotide encodingHGF and/or a polynucleotide encoding VEGF in the form of HVJ-liposomesby direct injection into the subarachnoid space of said subject therebypromoting said cerebral angiogenesis.
 4. A method of suppressingneuronal death in the brain comprising introducing into a human subjecta polynucleotide encoding HGF in the form of HVJ-liposomes by directinjection into the subarachnoid space of said subject therebysuppressing said neuronal death in the brain.
 5. A method of suppressingapoptosis of nerve cells in the brain comprising introducing into ahuman subject a polynucleotide encoding HGF in the form of HVJ-liposomesby direct injection into the subarachnoid space of said subject therebysuppressing said apoptosis of nerve cells.